Compositions and methods for simultaneous detection of volatile sulfur compounds and polyamines

ABSTRACT

Disclosed are compositions and methods useful for the rapid and facile simultaneous detection of malodorous bacterial metabolites in samples of expired breath and other fluids. The invention enables estimation, by simple visual inspection and comparison against standards, of the concentration of polyamines and volatile sulfur compounds in the micromolar to millimolar range.

FIELD OF THE INVENTION

The invention relates to, among other fields, the field of diagnostictests to detect the presence of pathogenic microorganisms.

BACKGROUND OF THE INVENTION

In normal circumstances many microorganisms live on or in humans withoutcausing harm to these humans. However, an increased number of certainmicroorganisms can be associated with a disease state. Particularlyproblematic is an increase in the number of microorganisms that producesignificant concentrations of chemicals that are toxic to humans. Suchtoxins include: volatile sulfur compounds (VSCs), such as hydrogensulfide, methyl mercaptan, dimethyl disulfide, and dimethyl sulfide; andpolyamines, such as putrescine and cadaverine. Higher concentrations ofvolatile sulfur compounds and of polyamines are associated with certaindisease states and with socially stigmatized conditions, such ashalitosis, or bad breath. It would be desirable to enable facilesimultaneous detection of volatile sulfur compounds and polyamines inorder to facilitate detection of the presence of potentially pathogenicmicroorganisms and of disease states and conditions with which thesemicroorganisms are associated.

The principal VSCs produced by oral bacteria are hydrogen sulfide (H₂S)and methylmercaptan (CH₃SH). H₂S is produced by the bacterial enzymeL-cysteine desulthydrase, which degrades the amino acid L-cysteine toproduce H₂S, ammonium and 2-ketopropanate. The other major VSC, CH₃SH,is produced by the action of the bacterial enzyme L-methionine gammalyase, which breaks down the amino acid L-methionine to produce CH₃SH,ammonium and 2-ketobutyrate. H₂S levels have been reported to occur atlevels as high as 2 mM in the gingival crevice of persons with severeperiodontal disease.

In addition to VSCs, oral bacteria have also been shown to produce highlevels of polyamines; chief among these are 1,4-diaminobutane(putrescine) and 1,5-diaminopentane (cadaverine). Putrescine is producedby the action of the bacterial enzyme ornithine decarboxylase, whichdegrades the amino acid L-ornithine to produce putrescine and carbondioxide. Cadaverine is produced by the bacterial enzyme lysinedecarboxylase, which breaks down the amino acid L-lysine to cadaverineand carbon dioxide.

Millimolar levels of these malodorous bacterial waste products have beenreported in the gingival crevicular fluid (GCF) of persons with severeperiodontal disease. Their presence imparts a foul odor to the breath ofpersons with the disease. In addition to being malodorous, VSCs havealso been shown to be toxic and adversely affect a number of mammalianenzymes. Production of these waste products by bacteria residing on thedorsum of the tongue imparts a foul odor to expired breath, resulting inhalitosis. Together these two classes of bacterial waste productscombine to impart a foul odor to the breath of persons with halitosisand periodontal disease. The levels of these waste products in GCFcorrelate directly with the level of periodontal disease activity. Thelevels of these waste products in expired air correlate directly withthe level of bad breath or halitosis.

SUMMARY OF THE INVENTION

Accordingly, the invention provides compositions and methods forsimultaneous detection of volatile sulfur compounds and polyamines.

The invention provides inter alia a chromogenic (calorimetric)biochemical assay to detect volatile sulfur compounds (VSCs) andpolyamines produced by oral bacteria residing on the dorsum of thetongue and in the gingival crevice.

The invention provides inter alia an assay to detect VSCs and polyaminesin saliva and expired breath.

In an aspect of the invention, the absorbance of a chromogeniccomposition according to the invention correlates directly with theconcentration of VSCs and polyamines in a fluid with which thecomposition is intermixed.

DETAILED DESCRIPTION OF THE INVENTION

At the outset of the description, it is helpful to note the meaning ofeach of several terms recurring throughout the specification and claims.

When used in connection with the invention, a “volatile sulfur compound”or “VSC” refers to a composition having a thiol or —SH functional groupor to a composition having a thiolate or sulfide anion. Examples of VSCsinclude hydrogen sulfide and methyl mercaptan (methanethiol).

When used in connection with the invention, “polyamine” has its usualmeaning in the biochemical arts and includes a composition of thegeneral formula NH₂RNH₂. Examples of polyamines include putrescine andcadaverine.

When used in connection with the invention, “DTNB” refers to5,5′-Dithiobis(2-nitrobenzoic acid). DTNB is also known as Ellman'sreagent and has CAS number 69-78-3.

When used in connection with the invention, “2-IT” refers to2-Iminothiolane. 2-Iminothiolane hydrochloride is known as Traut'sreagent and has CAS number 4781-83-3.

The invention provides inter alia a chromogenic (calorimetric)biochemical assay to detect volatile sulfur compounds (VSCs) andpolyamines produced by oral bacteria residing on the dorsum of thetongue and in the gingival crevice.

The invention provides inter alia an assay to detect VSCs and polyaminesin saliva and expired breath.

In an aspect of the invention, the absorbance of a chromogeniccomposition according to the invention correlates directly with theconcentration of VSCs and polyamines in a fluid with which thecomposition is intermixed.

In an embodiment, the invention provides a chemically stabilized,buffered composition comprising 5,5′-Dithiobis(2-nitrobenzoic acid)(DTNB) to detect VSCs and 2-Iminothiolane (2-IT) to detect polyamines.In this regard, DTNB and other so-called sulfhydryl reagents areexamples of VSC-reactive means, and 2-IT and other thiolating reagentswhich modify primary amino groups are examples of polyamine-reactivemeans.

In a particular embodiment, a composition according to the inventioncontains 0.5 millimolar (0.5 mM) DTNB and 0.5 mM 2-IT in 20 mM imidazoleor 20 mM sodium phosphate, pH 7.0 buffer containing 0.1 mM manganesechloride (MnCl₂). Such composition is referred to infra as “reagentmixture.” Without intending to be bound by theory, it is observed that,in such an embodiment, such concentration of MnCl₂ and such buffer andsuch pH are associated with particularly favorable stability, such thatthe shelf-life of the reagent mixture is 12 months or longer when storedat room temperature. A similar embodiment, in which a compositionaccording to the invention contains 0.1 mM cobalt chloride or 0.1 mMmagnesium chloride instead of the aforementioned 0.1 mM manganesechloride, also possesses particularly favorable stability. In thisregard, imidazole buffer around pH 7, phosphate buffer around pH 7,manganese chloride, magnesium chloride, and cobalt chloride are examplesof shelf-life-enhancing means. Theory notwithstanding, it is noted thatmagnesium is an alkaline earth metal and that managese and cobalt aretransition metals.

Without intending to be bound by theory, it is known in the art thatDTNB reacts with the sulfide or thiolate anion formed from the —SHfunctional group present in H₂S or CH₃SH. In such reaction, one of thedisulfide sulfurs of DTNB is attacked by the HS⁻ or CH₃S⁻ anion,whereupon the 5-thio-2-nitrobenzoate anion (TNB) is released. TNB existsin equilibrium with the tautomeric thioquinone. The thioquinone has anabsorption (absorbance) maximum at 412 nm with a molar extinctioncoefficient of 13,600 M⁻¹ cm⁻¹. This allows for the reaction to bequantified spectrophotometrically or visually by the increase in theyellow color of the mixture.

While in the art the calorimetric reaction of DTNB may be regarded asoptimally performed at pH 8.0, DTNB is unstable when stored for severaldays at room temperature under such conditions. It was unexpectedlyfound that the inclusion of metal salt and the adjustment of pH aroundneutrality impart the favorable property of a markedly extendedshelf-life. By way of example and not of limitation, it was found that aDTNB mixture was maintained stable for more than one year at roomtemperature when the mixture included phosphate or imidazole buffer, pH7.0, and 0.1 mM MnCl₂. Hence the invention provides inter alia achromogenic composition the absorbance of which correlates directly withthe concentration of VSCs in a fluid with which the composition isintermixed and which composition has a shelf-life of at least one year.

It is known in the art that 2-IT thiolates primary amines underappropriate conditions. A composition according to the invention isuseful for the detection of primary amines, including polyamines, suchas the malodorous diamines putrescine and cadaverine. When a compositionaccording to the invention is intermixed or otherwise contacted with asample that contains diamines, 2-IT of the composition reacts withprimary amino (—NH₂) groups of the sample's diamines to yield a reactionproduct with at least one free sulfhydryl group. Such a free sulfhydrylgroup, or its thiolate anion, reacts with DTNB of the reagent mixture,releasing the TNB anion and causing an increase in absorbance at 412 nm.

Compositions and methods according to the invention are useful fordetecting the presence of malodorous toxins produced by bacteriaresiding on the dorsum of the tongue and in GCF obtained from thegingival sulcus.

For example, in an aspect of a method according to the invention, tonguescrapings are obtained by gently rubbing the cotton tip of a sterile,cotton-tipped applicator against the dorsum of the tongue to form ascraping-laden applicator tip. The scraping-laden applicator tip is thencontacted for 2 minutes at room temperature with a volume of 0.1 ml ofreagent mixture contained within a sterile, screw-capped 1.5 mlmicrocentrifuge tube, forming a chromogen-developed applicator tip. Thecolor of the chromogen-developed applicator tip is compared to standardson a color-coded chart. The chart is scaled in shades of yellow from LOW(light yellow) corresponding to <0.2 mM H₂S to HIGH (bright yellow)corresponding to >2 mM H₂S.

Also by way of example, in an aspect of a method according to theinvention, for detecting the presence of VSCs and polyamines in GCF, aGCF sample is obtained by inserting a sterile paper absorbant point intothe gingival sulcus and waiting 1 minute for the GCF sample to absorb,thereby forming a GCF-laden paper point. The GCF-laden paper point isthen contacted for 5 minutes at room temperature with a volume of 0.1 mlof reagent mixture contained within a sterile, screw-capped 1.5 mlmicrocentrifuge tube, forming a chromogen-developed paper point. Thecolor of the chromogen-developed paper point is compared to a standardcolor chart calibrated linearly from 0 (none detectable) to 5 (extreme),corresponding to 2 mM hydrogen sulfide.

Also by way of example, in an aspect of a method according to theinvention, VSCs and polyamines are detected and quantifiedspectrophotometrically through use of the reagent mixture. In amicrocentrifuge tube, a GCF-laden paper point is submersed in 450microliters of 20 mM Tris-HCl, pH 7.75 buffer. When it is desired toperform a spectrophotometric assay according to the invention, a volumeof 50 microliters of a 10×concentrated reagent mixture in 50% DMSO, 50mM imidazole, pH 6.4 buffer containing 1.0 mM MnCl₂ is added to andintermixed with the contents of the microcentrifuge tube, and theresultant mixture is incubated at room temperature for 8 minutes.Afterwards, the incubated resultant mixture is transferred to asemi-micro 1.5 ml disposable plastic cuvet, for example by means of adisposable transfer pipette. The concentration of VSC and polyamines isdetermined by measuring the absorbance of the incubated resultantmixture through use of a spectrophotometer or colorimeter and bycomparison of the measured absorbance to a reference standard. Bymeasuring the absorbance of a negative control or a blank, backgroundabsorbance readings are obtained for use in comparison to the measuredabsorbance of the incubated resultant mixture.

A method of detection of VSCs and polyamines in GCF according to theinvention, such as described in either of the two immediately precedingparagraphs, is observed to possess a lower limit of detection of no morethan about 0.02 mM H₂S in a sample, and an upper limit of responsiverange near about 2 mM H₂S. If the sample contains polyamines butessentially no VSCs, this 0.02 mM lower limit corresponds to aconcentration of no more than about 0.02 mM polyamines in the sample. Ifthe sample contains VSCs but essentially no polyamines, this lower limitcorresponds to a concentration of no more than about 0.02 mM VSCs in thesample. For detection of polyamines and VSCs according to the invention,a mode of carrying out the invention whereby a composition according tothe invention is prepared using, as solvent, reagent grade nanopurewater, wherein concentrations of adventitious metals are extremely low,is in general to be preferred.

The invention also provides methods for detection of VSCs and polyaminesin saliva and expired breath.

For example, in an aspect of a method according to the invention, forsaliva testing, a filter paper disk is impregnated with the reagentmixture by contacting the disk with the reagent mixture, and allowingexcess solvent to evaporate, to form an impregnated disk. Theimpregnated disk is affixed to the bottom interior of a sterile,graduated specimen cup. A volume of saliva is then spit by a subjectonto the impregnated disk in the specimen cup. Reaction is allowed toproceed in and on the impregnated disk. After a fixed period of time,the concentration of VSCs and polyamines is determined by comparison ofthe color developed on the surface of the impregnated disk against astandard such as a scaled color chart.

For example, in an aspect of a method according to the invention, forbreath testing, a volume of 0.1 ml of reagent mixture is contacted witha filter paper disk, and excess solvent is allowed to evaporate, to forman impregnated disk. The impregnated disk is then affixed to the distalaperture of a cylindrical tube, said tube having an aperture at eachend, the proximal aperture being of such length and width, for example,that a subject might conveniently expire breath through the proximalaperture, and the distal aperture being of such length and width, forexample, that the impregnated disk covers the entire distal aperture. Asubject then expires breath into the tube through the proximal apertureof the tube and toward the distal aperture of the tube, at least aportion of the expired breath contacting the impregnated disk affixed tothe distal aperture of the tube. Reaction is allowed to proceed in andon the impregnated disk. After a fixed period of time, the concentrationof VSCs and polyamines is determined by comparison of the colordeveloped on the surface of the impregnated disk against a standard suchas a scaled color chart.

While filter paper is an exemplary matrix according to the invention,cotton is also a suitable matrix according to the invention, as is anyof a number of other inert matrices, such as those derived frompolysaccharides which are low in primary amino groups.

When DTNB and 2-IT are included in a MnCl₂-stabilized buffered mixtureaccording to the invention, the levels of two principal classes ofanaerobic bacterial waste products are conveniently detected andquantified either visually with a yellow color scale orspectrophotometrically with a colorimeter or spectrophotometer at 412nm. The simplicity of the use of compositions and methods according tothe invention facilitates in-office testing by health care professionalsand home testing by patients for halitosis and periodontal disease.Compositions and methods according to the invention thus are useful forthe detection of VSCs in samples comprising air, water, oil, or fluidsin contact with foodstuffs or foods, and hence are also useful for thedetection of products of microbial metabolism, pathogenicmicroorganisms, halitosis, VSC pollution, or food spoilage orcontamination.

The invention provides a diagnostic composition comprising 2-IT, DTNB, asolvent, a buffer, and a metal salt.

The invention provides a neutral diagnostic solution comprising fromabout 0.1 to about 2.5 millimoles of 2-IT per liter of the solution,from about 0.1 to about 2.5 millimoles of DTNB per liter of thesolution, from about 0.02 to about 0.5 millimoles of MnCl₂ or MgCl₂ orCoCl₂ per liter of the solution, and (a) from about 4 to about 100millimoles of imidazole per liter of the solution or (b) from about 4 toabout 100 millimoles of sodium phosphate per liter of the solution.

The invention provides a method of detecting the presence of a VSC or aprimary amine in a sample of a physiological fluid, said methodcomprising the step of contacting the sample with a compositioncomprising 2-IT, DTNB, a solvent, a buffer, and a metal salt.

The invention provides a method of detecting the presence of a VSC or aprimary amine in a sample of a physiological fluid, said methodcomprising the step of contacting the sample with a neutral diagnosticsolution comprising from about 0.1 to about 2.5 millimoles of 2-IT perliter of the solution, from about 0.1 to about 2.5 millimoles of DTNBper liter of the solution, from about 0.02 to about 0.5 millimoles ofMnCl₂ or MgCl₂ or CoCl₂ per liter of the solution, and (a) from about 4to about 100 millimoles of imidazole per liter of the solution or (b)from about 4 to about 100 millimoles of sodium phosphate per liter ofthe solution.

The invention provides a chromogen, the color of which chromogendevelops more rapidly after the chromogen is contacted with a firstmixture comprising at least 20 micromolar polyamine or at least 20micromolar VSC than the color develops in a second mixture identical incomposition to the first mixture except that the second mixturecomprises no more than 1 micromolar polyamine and no more than 1micromolar VSC.

The invention provides a chromogen, the color of which chromogendevelops more rapidly after the chromogen is contacted with a firstmixture comprising at least 20 micromolar polyamine or at least 20micromolar VSC than the color develops in a second mixture identical incomposition to the first mixture except that the second mixturecomprises no more than 1 micromolar polyamine and no more than 1micromolar VSC, wherein a neutral solution of the chromogen has ashelf-life of at least one year.

The invention provides a chromogen, the color of which chromogendevelops more rapidly after the chromogen is contacted with a firstmixture comprising at least 20 micromolar polyamine or at least 20micromolar VSC than the color develops in a second mixture identical incomposition to the first mixture except that the second mixturecomprises no more than 1 micromolar polyamine and no more than 1micromolar VSC, wherein the color of the developed chromogen is yellow.

The invention provides a chromogen, the color of which chromogendevelops more rapidly after the chromogen is contacted with a firstmixture comprising at least 20 micromolar polyamine or at least 20micromolar VSC than the color develops in a second mixture identical incomposition to the first mixture except that the second mixturecomprises no more than 1 micromolar polyamine and no more than 1micromolar VSC, wherein the chromogen comprises 2-IT and DTNB.

The invention provides a test kit comprising a chromogen, the color ofwhich chromogen develops more rapidly after the chromogen is contactedwith a first mixture comprising at least 20 micromolar polyamine or atleast 20 micromolar VSC than the color develops in a second mixtureidentical in composition to the first mixture except that the secondmixture comprises no more than 1 micromolar polyamine and no more than 1micromolar VSC.

The invention provides a test kit comprising a chromogen, the color ofwhich chromogen develops more rapidly after the chromogen is contactedwith a first mixture comprising at least 20 micromolar polyamine or atleast 20 micromolar VSC than the color develops in a second mixtureidentical in composition to the first mixture except that the secondmixture comprises no more than 1 micromolar polyamine and no more than 1micromolar VSC, wherein the test kit has a shelf-life of at least oneyear.

The invention provides a method of detecting the presence of amicroorganism in a sample of a physiological fluid, said methodcomprising contacting the sample with a chromogen, the color of whichchromogen develops more rapidly after the chromogen is contacted with afirst mixture comprising at least 20 micromolar polyamine or at least 20micromolar VSC than the color develops in a second mixture identical incomposition to the first mixture except that the second mixturecomprises no more than 1 micromolar polyamine and no more than 1micromolar VSC.

The invention provides a method of detecting the presence of amicroorganism in a sample of a physiological fluid, said methodcomprising contacting the sample with a chromogen, the color of whichchromogen develops more rapidly after the chromogen is contacted with afirst mixture comprising at least 20 micromolar polyamine or at least 20micromolar VSC than the color develops in a second mixture identical incomposition to the first mixture that the second mixture comprises nomore than 1 micromolar polyamine and no more than 1 micromolar VSC,wherein the microorganism is an anaerobe or a facultative aerobe.

The invention provides a method of detecting the presence of amicroorganism in a sample of a physiological fluid, said methodcomprising contacting the sample with a chromogen, the color of whichchromogen develops more rapidly after the chromogen is contacted with afirst mixture comprising at least 20 micromolar polyamine or at least 20micromolar VSC than the color develops in a second mixture identical incomposition to the first mixture except that the second mixturecomprises no more than 1 micromolar polyamine and no more than 1micromolar VSC, wherein the physiological fluid comprises GCF, saliva,tongue scrapings, or expired breath.

It will therefore be readily understood by those persons skilled in theart that the invention is susceptible of a broad utility andapplication. Many embodiments and adaptations of the invention otherthan those herein described, as well as many variations, modificationsand equivalent arrangements, will be apparent from or reasonablysuggested by the present invention and the foregoing descriptionthereof, without departing from the substance or scope of the presentinvention. Accordingly, while the invention has been described herein indetail in relation to its preferred embodiment, it is to be understoodthat this disclosure is only illustrative and exemplary of the inventionand is made merely for purposes of providing a full and enablingdisclosure of the invention. The foregoing disclosure is not intended orto be construed to limit the invention or otherwise to exclude any suchother embodiments, adaptations, variations, modifications or equivalentarrangements, the invention being limited only by the claims appendedhereto and the equivalents thereof.

1. A test kit useful for the detection of a plurality of microbialmetabolites in a fluid, the test kit comprising: (a) a detection matrixcomprising a product of a process whereby an inert matrix is contactedwith an aqueous chromogenic composition, the aqueous chromogeniccomposition comprising (i) water as solvent, (ii) a first detectionreagent that reacts with a polyamine to form a VSC, (iii) a seconddetection reagent that reacts with a VSC to form a visible product, saidvisible product possessing an absorption maximum in the visible portionof the electromagnetic spectrum, and (iv) a shelf-life-enhancingreagent, said shelf-life-enhancing reagent being an alkaline earth metalor a transition metal; and (b) a color standard for comparison to colordeveloped on the detection matrix; wherein (I) the plurality ofmicrobial metabolites comprises at least one polyamine and at least oneVSC; (II) the fluid comprises GCF and/or saliva; (III) the absorbance ofthe aqueous chromogenic composition correlates directly with theconcentration of VSCs and polyamines in a fluid sample with which theaqueous chromogenic composition is intermixed; (IV) the color of theaqueous chromogenic composition develops more rapidly after the aqueouschromogenic composition is contacted with a first fluid samplecontaining at least 20 micromolar polyamine or at least 20 micromolarVSC than the color of the aqueous chromogenic composition develops afterthe aqueous chromogenic composition is contacted with a second fluidsample identical to the first fluid sample except that the second fluidsample contains no more than 1 micromolar polyamine and no more than 1micromolar VSC; and (V) the shelf-life of the test kit is at least oneyear.